Adenoviral systems and the uses thereof

ABSTRACT

The invention is related to a nucleic acid comprising an adenoviral nucleic acid, which also comprises a nucleic acid sequence coding for YB-1.

The present invention is related to nucleic acids comprising adenoviralnucleic acid, adenoviruses comprising these nucleic acids and the usethereof.

Numerous therapeutical plans are at present used to treat tumours. Inaddition to the use of surgical techniques, chemotherapy and radiationtherapy are mainly used. However, all these methods are associated withconsiderable side-effects for the patient.

The use of replication-selective oncolytic viruses has created a newplatform for treating tumours. In this method a selective intratumoralreplication of a viral agent is induced which results in virusreplication, lysis of the infected tumour cell and spread of the virusto the neighbouring tumour cells. Since the virus is only able toreplicate in tumour cells, normal tissue is protected from infection bythe virus and thus from viral lysis. Examples of suchreplication-selective oncolytic viruses are gene attenuated adenovirusand herpes viruses (Martuza, R. et al. Science 252, 854-858 (1991);Fueyo, J. et al. Oncogene 19, 2-12 (2000)).

An example of such an adenovirus is dl 1520 (Onyx-015) which has alreadybeen successfully used in clinical phases I and II (Khuri, F. et al.Nature Medicine 6, 879-885 (2000)). Onyx-015 is an adenovirus in whichthe E1B 55 kDa gene is deleted. The E1B 55 kDa gene product is involvedin the inhibition of p53, the transport of viral mRNA and switching offprotein synthesis of the host cell. In this connection p53 is inhibitedby formation of a complex consisting of p53 and the E1B 55 kDa proteincoded by the adenovirus. P53 which is coded by TP53 is the basis for acomplex regulatory mechanism (Zambetti, G. P. et al., FASEB J. 7,855-865) which, among others, results in suppression of the efficientreplication of viruses such as adenoviruses in the cell. The gene TP53is deleted or mutated in about 50% of all human tumours and consequentlythe desired apoptosis does not occur as a result of chemotherapy orradiation therapy and thus these tumour treatments are usuallyunsuccessful.

DNA tumour viruses such as adenoviruses drive the infected cells intothe S phase of the cell cycle in order to facilitate viral DNAreplication. Onyx-015 does not express the E1B 55 kDa protein andreplicates selectively in tumour cells but not in normal cells.Furthermore another selectivity is that tumours that are deficient inp53 are necrosed by the viral lysis of the tumour cells to a greaterextent than those tumours which have the p53 wild-type (Khuri et al.,loc.cit.). Despite the fundamental effectiveness of Onyx-015 inviral-induced oncolysis in the case of p53-deficient tumours, thesuccess rate of 15% of treated tumours is very low.

Ries et al. (Ries, S. J. et al. Nature Medicine 6, 1128-1132 (2000))have described a basic approach for successfully using Onyx-015 even fortumours with the p53 wild-type. In this case the tumour suppressorprotein p14ARF is not expressed. As a result of the absence of p14ARFthe normal reaction of the p53 system to a viral infection does not takeplace and thus allows Onyx-015 to also replicate in these tumours.However, an application of this finding requires that there is asuitable genetic background in the tumour cell or that such a backgroundis provided by suitable therapeutic measures. In the former case thiswould further reduce the number of tumours that can be treated byOnyx-015 and in the latter case it would require a complicated change inthe genetic background of the tumour cells.

It is an object of the present invention to improve the existingadenoviral systems for viral-induced oncolysis. In particular the aim isto increase the success rate of tumour treatment compared with the priorart.

Another object of the present invention is to provide adenoviral systemsfor viral-induced oncolysis which are also effective on tumours that areof the p53 wild-type.

According to the invention the object is achieved in a first aspect by anucleic acid comprising an adenoviral nucleic acid wherein the nucleicacid comprises a nucleic acid sequencing coding for YB-1.

One embodiment provides that the adenoviral nucleic acid comprises anucleic acid coding for E1-B.

Another embodiment provides that the adenoviral nucleic acid comprises anucleic acid coding for E4orf6.

In yet another embodiment a promoter is provided which controls theexpression of E1-B.

In another embodiment a promoter is provided which controls theexpression of E4orf6.

Another embodiment provides that E1B is the E1-B 55 kDa protein.

Another embodiment provides that the nucleic acid codes for functionallyinactive gene products E1B and/or E1 and/or E3 and/or E4.

In a second aspect the object is achieved by a nucleic acid comprising anucleic acid sequence coding for YB-1 and a nucleic acid sequencemediating a nuclear transport of YB-1.

Another embodiment provides that the nucleic acid sequence mediating thenuclear transport of YB-1 is selected from the group comprising signalsequences and transport sequences.

A third aspect of the invention is related to a nucleic acid comprisingan adenoviral nucleic acid wherein the adenoviral nucleic acid comprisesa tumour-specific promoter instead of a or the functional E2 latepromoter, in particular the functionally active E2 late promoter.

A fourth aspect of the invention concerns a nucleic acid comprising anadenoviral nucleic acid wherein the adenoviral nucleic acid comprises atissue-specific promoter instead of a or the functional E2 latepromoter, in particular the functionally active E2 late promoter.

The object is achieved in a fifth aspect by an adenoviral replicationsystem comprising an adenoviral nucleic acid, whereby the adenoviralnucleic acid is deficient for the expression of the E1A protein, andcomprising a nucleic acid of a helper virus, whereby the nucleic acid ofthe helper virus comprises a nucleic acid sequence which codes for YB-1.

One embodiment provides that the adenoviral nucleic acid and/or thenucleic acid of the helper virus is present as a replicable vector.

The object is achieved in a sixth aspect by a vector, preferably anexpression vector, which comprises one of the nucleic acids according tothe present invention.

The object is achieved in a seventh aspect by a group of vectorscomprising of at least two vectors, whereby the overall vector groupcontains an adenoviral replication system according to the presentinvention.

One embodiment provides that each component of the adenoviralreplication system is located on a suitable vector, preferably anexpression vector.

Another embodiment provides that at least two components of theadenoviral replication system are located on a vector of the group ofvectors.

An eighth aspect of the invention is related to an adenovirus comprisingone of the nucleic acids according to the present invention.

One embodiment provides that the adenovirus comprises a capsid.

A ninth aspect of the invention is related to a cell comprising anucleic acid according to the present invention and/or an adenoviralreplication system according to the present invention and/or a vectoraccording to the present invention and/or a group of vectors accordingto the present invention and/or an adenovirus according to the presentinvention.

A tenth aspect of the invention is related to the use of a nucleic acidaccording to the present invention and/or an adenoviral replicationsystem according to the present invention and/or a vector according tothe present invention and/or a group of vectors according to the presentinvention and/or an adenovirus according to the present invention and/ora cell according to the present invention for the manufacture of amedicament.

One embodiment provides that the medicament is used for the treatmentand/or prophylaxis of tumour diseases.

In yet another embodiment the medicament additionally comprises apharmaceutically effective compound.

One embodiment provides that the pharmaceutically effective compound isselected from the group comprising cytostatic agents. Suitablecytostatic agents are among others cis-platinum, Taxol, Daunoblastin,Adriamycin and Mitoxantron.

An eleventh aspect of the invention is related to the use of a nucleicacid according to the present invention and/or an adenoviral replicationsystem according to the present invention and/or a vector according tothe present invention and/or a group of vectors according to the presentinvention and/or an adenovirus according to the present invention and/ora cell according to the present invention for the manufacture of amedicament for the treatment and/or prophylaxis of tumour diseases,whereby YB-1 is located in the nucleus of the tumour cells independentof the cell cycle.

One embodiment provides that the nucleic acid codes for an adenoviralnucleic acid and that the adenoviral nucleic acid is E1B-deficient, andin particular E1B 55 kDa-deficient.

Another embodiment provides that the adenovirus is E1B-deficient, and inparticular E1B 55 kDa-deficient.

An alternative embodiment provides that the nucleic acid codes for anadenoviral nucleic acid and that the adenoviral nucleic acid codes forE1B, and in particular for E1B 55 kDa.

One embodiment provides that the adenovirus expresses E1B, and inparticular E1B 55 kDa.

Yet a further embodiment provides that the adenoviral nucleic acid codesfor E1A.

Yet another embodiment provides that the adenovirus expresses E1A.

In the various embodiments the tumour and/or the tumour disease isselected from the group comprising p53-positive tumours, p53-negativetumours, malignant tumours, benign tumours and combinations thereof.

A twelfth aspect of the invention is related to a method for thescreening of patients which can be treated with an E1B-deficient,preferably E1B 55 kDa-deficient adenovirus which comprises the followingsteps:

-   -   examining a sample of the tumour tissue and    -   determining whether YB-1 is located in the nucleus independently        of the cell cycle.

One embodiment provides that the tumour tissue is examined using anagent which is selected from the group comprising antibodies againstYB-1.

A thirteenth aspect of the invention is related to the use of anantibody against YB-1 to determine which patients, in particular whichtumour patients can be treated with an E1B-deficient adenovirus,preferably an E1B 55 kDa-deficient adenovirus.

A fourteenth aspect of the invention is related to a complex comprisingat least one YB-1 molecule and at least one E1B 55 kDa protein.

One embodiment provides that the YB-1 molecule is a transgenic YB-1molecule.

Another embodiment provides that the YB-1 is YB-1 expressed in thenucleus.

In connection with the nucleic acids disclosed herein, the term is alsoused in the sense of nucleic acid sequences. The nucleic acids accordingto the present invention and the adenoviruses according to the presentinvention are preferably recombinant products especially when they havebeen changed compared to the wild-type. In the context of the presentinvention the nucleic acids according to the present invention and theadenoviruses according to the present invention typically have an E1deletion, an E1-E3 deletion and/or an E4 deletion i.e. the nucleic acidor the corresponding adenoviruses are not able to produce functionallyactive E1 and/or E3 and/or E4 expression products or such productscannot be produced from them. This is typically achieved by a deletionor an appropriate mutation, including a point mutation.

A fifteenth aspect of the present invention is related to the use of acomplex comprising at least one YB-1 molecule and at least one E1Aprotein.

One embodiment provides that one or more of the proteins of the E1Aregion have a transactivating effect on adenoviral gene expression butdoes/do not activate the replication of an adenovirus.

A sixteenth aspect of the invention is related to the use of a complexaccording to the present invention comprising at least one YB-1 moleculeand at least one E1A protein, for treating tumours, and in particularfor tumour lysis.

A seventeenth aspect of the present invention is related to the use ofan E1B-deficient adenovirus for the manufacture of a medicament fortreating tumours which have YB-1 in the nucleus.

A preferred embodiment provides that the nuclear localization of YB-1 isachieved under the influence of exogenous measures.

A particularly preferred embodiment provides that the exogenous measuresare measures which are selected from the group comprising irradiation,cytostatic agents and hyperthermia.

Yet a further aspect of the invention provides that the exogenousmeasure is used on the organism for which the medicament is to be used.

Other embodiments are derived from the subclaims.

The present invention is based on the surprising finding that afterinfection of a cell and typically of a tumour cell with an adenovirus acomplex is formed between YB-1 and the adenoviral gene product E1B-55kDa and this complex formation results in a transport of YB-1 into thenucleus which allows an effective replication of the virus in the cellnucleus in vivo. It was also found that E4orf6 also binds to E1B-55 K(Weigel, S., Dobbelstein, M. J. Virology, 74, 764-772, 2000). Thenuclear export signal within the E4orf6 protein of adenovirus type 5supports virus replication and cytoplasmic accumulation of viral mRNA;(Keith N. Leppard, Seminars in Virology, 8, 301-307, 1998. Regulated RNAprocessing and RNA transport during adenovirus infection) and thusmediates transport or distribution of E1B-55 K in the nucleus. Hence theinteraction between E1B-55 K and YB-1 or E1B-55 K, YB-1 and E4orf6results in an efficient replication of the virus which in turn leads toa lysis of the cell, release of the virus and infection and lysis ofneighbouring cells so that if a tumour cell or a tumour is infected, thetumour is ultimately lysed, i.e. an oncolysis occurs.

Another finding on which the present invention is based, is that YB-1binds as a transcription factor to the late E2 promoter of adenovirusand as a result activates the replication of the adenovirus. Thisprovides new adenoviruses and adenoviral systems for oncolysis.

The present invention is also based on the surprising finding that theexpression of the transgene YB-1 in an adenoviral vector leads to viralreplication. The viral genes E1B, E3 and E4 are not switched on in thisprocess which is mainly due to the fact that E1 and/or E3 are deleted inthe adenoviral vector. However, these genes are necessary for a veryefficient replication and particle formation (Goodrum, F. D., Omelles,D. A. Roles for the E4orf6, orf3 and E1B 55-kilodalton proteins in cellcycle-independent adenovirus replication. J. Virol. 73, 74474-7488(1999); Medghalchi, S., Padmanabhan, R, Ketner, G. Early region 4modulates adenovirus DNA replication by two genetically separablemechanisms. Virology, 236, 8-17 (1997). It is also known that twoproteins (the 12S and the 13S protein) which are coded by E1A, controland induce the expression of other adenoviral genes (Nevins, J. R.Mechanism of activation of early viral transcription by the adenovirusE1 A gene products. Cell 26, 213-220 (1981); Boulanger, P et al. (1991);Biochem. J. 275, 281-299). It has been shown that the CR3 region of the13S protein is mainly responsible for the transactivating function (WongH K, Ziff E B. Complementary functions of E1a conserved region 1cooperate with conserved region 3 to activate adenovirus serotype 5early promoters. J. Virol. 1994, 68(8):4910-20). Adenoviruses withcertain deletions in the CR1 and/or CR2 region of the 13S protein cannotreplicate but still have a transactivating effect on the viral genes andpromoters (Wong H K, Ziff E B. Complementary functions of E1a conservedregion 1 cooperate with conserved region 3 to activate adenovirusserotype 5 early promoters. J. Virol. 1994, 69(8):4910-20).

A combination of such a system, i.e. a system which switches on theviral genes but is not capable of viral replication, with a tumour ortissue-specific expression of the transgene YB-1 would, in contrast,allow a very effective viral replication or particle formation and thusoncolysis. Any of the promoters that have been described herein anywherecan be used as suitable tumour-specific or tissue-specific promoters.

YB-1 is a representative of the Y box protein family which binds to theDNA Y box sequence motif. The Y box motif is a transcriptionalregulatory element which is found in the promoter or enhancer regions ofa number of different genes which play a role in the regulation of cellproliferation (Ladomery, M. et al., 1995; Bioassays 17:9-11; Didier, D.K. et al., 1988, PNAS, 85, 7322-7326).

Adenoviruses are known in the prior art. They are dsDNA viruses(Boulanger, P. et al. (1991); Biochem. J. 275, 281-299). Theorganization of the genome is shown in FIG. 1. The complete nucleotidesequence of the adenoviral genome is known and is described inChroboczek, J. et al. (Chroboczek, J. et al., Virology 1992, 186,280-285). A part of the genome which is particularly important for theapplication of adenoviruses are the so-called early genes and their geneproducts which are referred to as E1, E2, E3 and E4. E1 consists of twogene products E1 A and E1B which represent oncogenes. The total of threegene products of the E2 group are involved in replication together withthe gene products E3 and E4.

The adenoviral systems known in the prior art for oncolysis such asOnyx-015 have an E1B-55 kDa protein deletion. This deletion was madeunder the assumption that an intact p53 gene counteracts an efficientreplication in vivo and in order to ensure an adenoviral replication invivo only in p53-negative/mutated cells, but results in a reduction bytwo orders of magnitude in the particle number compared to the wild-typeas a result of impaired replication. On the other hand these adenoviralsystems of the prior art rely on E1A in order to control in vivoreplication mediated by the E2 early promoter.

The present invention differs from this principle in that the adenoviralsystems described herein are based on the E2 late promoter.

Within the scope of the present invention the terms adenovirus andadenoviral systems are to be understood as having essentially the samemeaning. Adenovirus is to be understood especially as the complete virusparticle containing the capsid and nucleic acid. The term adenoviralsystem indicates in particular that the nucleic acid is changed comparedto the wild-type. Such changes preferably comprise changes in thestructure of the genome of the adenovirus such as the deletion and/oraddition and/or mutation of promoters, regulatory sequences and codingsequences (such as the reading frame). The term adenoviral systems isalso preferably used in the context of a vector.

The adenoviral nucleic acids to which reference is made herein, areknown in the prior art. A person skilled in this field will know how todelete or mutate the adenoviral nucleic acid sequences that areunimportant for the invention. Such deletions can for example affect thenucleic acid coding for E3. In preferred embodiments these adenoviralnucleic acids can still be packaged in the viral capsid and thus forminfectious particles. The same applies to the nucleic acids according tothe present invention. In general it should also be noted that theadenoviral systems can be deficient with regard to one or moreexpression products. It should be taken into consideration that this maybe due to the fact that the nucleic acid coding for the expressionproduct is mutated or deleted completely or to such an extent thatessentially no more expression product is formed or that the regulatoryelements or the elements controlling expression such as promoters ortranscription factors, are absent whether at a nucleic acid level(absence of a promoter; cis-acting element) or at the level of thetranslation or transcription system (trans-acting elements). Inparticular the latter aspect may depend on the respective cellularbackground.

The nucleic acid according to the present invention which comprises anadenoviral nucleic acid and additionally a nucleic acid sequence codingfor YB-1, is preferably a recombinant nucleic acid. In this context thereading frame for the nucleic acid coding for YB-1 may be under thecontrol of an element controlling expression and/or translation. Thismay for example be an adenoviral promoter or a non-adenoviral promoter.Suitable non-adenoviral promoters can be selected from the groupcomprising cytomegalovirus promoter, RSV (Rous sarcoma virus) promoter,adenovirus-based promoter Va I and a non-viral YB-1 promoter. Otherviral promoters which can be used in connection with any aspects of theinvention disclosed herein are the telomerase promoter, the alphafetoprotein (AFP) promoter, the carcinoembryonic antigen promoter (CEA)(Cao, G., Kuriyama, S., Gao, J., Mitoro, A., Cui, L., Nakatani, T.,Zhang, X., Kikukawa, M., Pan, X., Fukui, H., i, Z. Comparison ofcarcinoembryonic antigen promoter regions isolated from human colorectalcarcinoma and normal adjacent mucosa to induce strong tumour-selectivegene expression. Int. J. Cancer, 78, 242-247, 1998), the L-plastinpromoter (Chung, I., Schwartz, P E., Crystal, R C., Pizzorno, G.Leavitt, J., Deisseroth, A B. Use of L-plastin promoter to develop anadenoviral system that confers transgene expression in ovarian cancercells but not in normal mesothelial cells. Cancer Gene Therapy, 6,99-106, 1999), arginine-vasopressin promoter (Coulson, J M, Staley, J.,Woll, P J. Tumour-specific arginine vasopressin promoter activation insmall cell lung cancer. British J. Cancer, 80, 1935-1944, 1999) and thePSA promoter (Hallenbeck P L, Chang, Y N, Hay, C. Golightly, D.,Stewart, D., Lin, J., Phipps, S., Chiang, Y L. A novel tumour-specificreplication-restricted adenoviral vector for gene therapy ofhepatocellular carcinoma. Human Gene Therapy, 10, 1721-1733, 1999).

It is known that the telomerase promoter is of pivotal, importance inhuman cells. Thus telomerase activity is regulated by thetranscriptional control of the telomerase reverse transcriptase gene(hTERT) which represents the catalytic subunit of the enzyme. Telomeraseexpression is active in 85% of human tumour cells. In contrast it isinactive in most normal cells. Exceptions are germ cells and embryonictissue [Braunstein, I. et al. (2001). Human telomerase reversetranscription promoter regulation in normal and malignant human ovarianepithelial cells. Cancer Research, 61, 5529-5536; Majumdar AS et al.(2001). The telomerase reverse transcriptase promoter drives efficacioustumour suicide gene therapy while preventing hepatotoxicity encounteredwith constitutive promoters. Gene Therapy, 8, 568-578]. Detailedexaminations of the hTERT promoter have shown that fragments of thepromoter at a distance of 283 by and 82 by from the initiation codon aresufficient for a specific expression in tumour cells (Braunstein, I. etal.; Majumdar A S et al., loc.cit.). Hence this promoter and thespecific fragments are suitable for achieving a specific expression of atransgene only in tumour cells. The promoter should enable theexpression of the transgene YB-1 and/or a truncated form which is stillfunctionally active only in tumour cells. The expression of thetransgene in an adenoviral vector then leads to viral replication of theadenoviral vector and consequently to oncolysis. It is also within thescope of the present invention that the reading frame of YB-1 is inframe with one or more of the gene products of the adenoviral system.However, the reading frame of YB-1 may also be independent thereof. Thenucleic acid coding for YB-1 can be a complete sequence. However, it isalso possible within the scope of the present invention that the nucleicacid sequence is truncated. Such a truncated nucleic acid sequence andalso such a truncated YB-1 protein is especially within the scope of thepresent invention when such a truncated YB-1 still has a function orproperty like that of the complete YB-1. Such a function or property isfor example the ability to enter the nucleus, with or without binding tothe E1B-55 kDa protein, or to bind to the E2 late promoter.

The adenoviral nucleic acid which is comprised in the nucleic acidaccording to the present invention can be any adenoviral nucleic acidwhich itself leads to a replication event or leads to such a replicationevent in conjunction with other nucleic acid sequences. The othernucleic acid sequences can for example be YB-1. As elucidated below itis possible in this context that the sequences and/or gene productsrequired for replication are provided by helper viruses. An example ofsuch an adenoviral nucleic acid is the nucleic acid of Onyx-015 whichallows expression of E1A but not of E1B.

In the embodiment of the nucleic acid according to the present inventionwhich comprises a nucleic acid sequence coding for YB-1, the adenoviralnucleic acid may comprise a nucleic acid coding for E1-B. In thiscontext it is possible that although the nucleic acid coding for E1-B ispresent, the E1-B which it encodes, is not expressed. This can forexample be achieved when the nucleic acid coding for E1-B lacks asuitable promoter. This is for example the case when E1A is notexpressed. However, it is also within the scope of the present inventionthat E1-B is expressed, for example when the nucleic acid coding forE1-B is under the control of a suitable promoter. A suitable promoter isfor example selected from the group comprising the cytomegaloviruspromoter, RSV (Rous sarcoma virus) promoter, adenovirus-based promoterVa I and the non-viral YB-1 promoter. Moreover, the above-mentionedpromoters are also suitable in this case, i.e. the telomerase promoter,the alpha fetoprotein (AFP) promoter, the carcinoembryonic antigenpromoter (CEA), the L-plastin promoter, the arginine-vasopressinpromoter and the PSA promoter.

As disclosed herein E1-B-55 k is involved in the distribution and thetransport of YB-1 in the cell nucleus. Since E4orf6 in turn binds toE1-B-55k and is also responsible for the transport of E1-B-55 into thenucleus, E4orf6 also plays an important role in the distribution andtransport of YB-1 into the cell nucleus. Hence it is also within thescope of the present invention that the genes of the E4 region, and inparticular E4orf6, are also under the control of one of the promotersmentioned above. In this context a preferred embodiment provides thatthe adenoviral E4 promoter is no longer functional. It is particularlypreferred when the adenoviral E4 promoter is deleted.

One embodiment of the nucleic acid according to the present inventionwhich comprises a nucleic acid sequence coding for YB-1, provides thatthe adenoviral nucleic acid comprises a nucleic acid coding for E1-A butthat the adenoviral nucleic acid does not allow expression of E1-B andthus corresponds to the structure of the DNA of Onyx-015.

In general when reference is made herein to E1-B it preferably appliesto the E1 B 55 kDa protein unless stated otherwise.

If reference is made herein to coding nucleic acid sequences and theseare nucleic acid sequences that are known, it is within the scope of theinvention to not only use an identical sequence but also sequences thatare derived therefrom. Derived sequences are understood hereinespecially as sequences which still result in a gene product which has afunction that corresponds to a function of the non-derived sequence.This can be ascertained by simple routine tests. An example of suchderived nucleic acid sequences are nucleic acid sequences which code forthe same gene product in particular for the same amino acid sequence,but have a different base sequence as a result of the degeneracy of thegenetic code.

Another aspect of the nucleic acid according to the present inventioncomprising a nucleic acid coding for YB-1 and a nucleic acid sequencemediating transport of YB-1 into the nucleus, is based on the surprisingfinding that when YB-1 is present in the nucleus, especially independentof the cell cycle, an adenoviral replication occurs in the cell,preferably in a tumour cell. In connection therewith the nucleic acids,adenoviruses and adenoviral systems each according to the presentinvention can be used as such or in combination with the adenovirusesknown in the art such as Onyx-015, as adenoviruses and adenoviralsystems, respectively, and the corresponding nucleic acids therewith.

Suitable nucleic acid sequences for mediating nuclear transport areknown to the ones skilled in the art and are described for example in(Whittaker, G. R. et al., Virology, 246, 1-23, 1998; Friedberg, E. C.,TIBS 17, 347 1992; Jans, D. A. et al. Bioessays 2000 June; 22(6):532-44; Yoneda, Y., J. Biochem. (Tokyo) 1997 May; 121(5): 811-7;Boulikas, T., Crit. Rev. Eukaryot. Gene Expr. 1993; 3(3): 193-227).Different mechanisms can be used by the nucleic acid sequences mediatingnuclear transport. One of these mechanisms is that YB-1 is formed as afusion protein with a signal peptide and because of the signal peptideYB-1 is transported into the cell nucleus. Another mechanism is thatYB-1 is provided with a transport sequence which results in YB-1 beingtransported into the cell nucleus where it promotes viral replicationpreferably after it has been synthesized in the cytoplasm. An example ofa nucleic acid sequence that is particularly effective in mediatingtransport into the nucleus is the TAT sequence of HIV which, in additionto other suitable nucleic acid sequences of this type, is described forexample in Efthymiadis, A., Briggs, L J, Jans, DA. The HIV-1 thatnuclear localisation sequence confers novel nuclear import properties.JBC, 273, 1623.

Another aspect of the invention comprising a nucleic acid comprising anadenoviral nucleic acid whereby the adenoviral nucleic acid comprises atumour-specific promoter instead of the E2 late promoter, is also basedon the surprising finding that the expression and hence the replicationof the adenovirus and thus oncolysis depends essentially on the controlof the adenoviral genes and gene products, in particular in the E2region by the E2 late promoter, especially in vivo. The E2 region ofadenovirus which represents a transcription unit, is composed of the E2Aand E2B genes which code for proteins that are vital for viralreplication: pTP (precursor terminal protein), DNA polymerase and DBPwhich is a multifunctional DNA binding protein. Within the scope of thepresent invention the terms deleted promoter, non-functionally active,functionally non-active or non-functional promoter mean that thepromoter as such is no longer active, i.e. no longer results intranscription. Such a non-functional promoter can be generated by knownmethods by a person skilled in this art. For example this can beaccomplished by a complete deletion of the promoter, by a partialdeletion of the promoter or by a point mutation. Other methods ofmutating such promoters are, for example, to change the spatialrelationship between the elements of which the promoter is composedresulting in a functional inactivation.

The inactivation of the E2 late promoter and its replacement by atumour-specific or tissue-specific promoter and thus the production ofone of the nucleic acids according to the present invention, ensuresthat genes and gene products that are important for adenoviralreplication are under the control of the tumour or of the correspondingtissue and consequently replication of the adenovirus occursspecifically in the tumour or in a particular tissue. This meets therequirement for a specific virus-mediated lysis of cells and thusensures the safety of the system. In this connection the E2 latepromoter can be inactivated by being completely deleted. However, it isalso possible within the scope of the invention for the E2 late promoterto be changed in such a manner that it is no longer functionally activeas a promoter. This can for example be carried out by changing, e.g.mutating or deleting, the binding site of the promoter for YB-1 in sucha manner that YB-1 can no longer bind to the promoter. The deletion ofthe Y box from the promoter is an example of such a deletion. The termused herein that a nucleic acid according to the present inventioncomprises a tissue-specific or tumour-specific promoter instead of theE2 promoter encompasses the methods described above. To this extent theterm is to be understood in a functional manner.

In order to further improve the safety of the system which controls theexpression of the E2 gene products via a tumour-specific ortissue-specific promoter, in a preferred embodiment the E2 earlypromoter should be functionally inactive at the same time as the E2 latepromoter, for example deleted or altered in such a manner that it is nolonger functionally active as a promoter. This ensures that the E2 earlypromoter has no influence on the expression of the E2 genes. Instead thetwo promoters, i.e. the E2 late promoter and the E2 early promoter, arepreferably replaced by a tissue-specific or tumour-specific promoteraccording to the present invention. Also in this case the promotersdescribed herein in connection with the YB-1 encoding nucleic acidsequences can be used.

The adenoviral nucleic acid or the corresponding adenoviruses preferablycomprise the genes for E1A, E1B, E2 and E3. In preferred embodiments thenucleic acid coding for E3 can be deleted.

The adenovirus constructs described above and in particular theirnucleic acids can also be introduced in parts into a cell, in particulara tumour cell, in which case they interact due to the presence of thevarious individual components in such a manner as if the individualcomponents were derived from an individual nucleic acid. A typicalexample of this which is referred to herein as an adenoviral replicationsystem, provides that the adenoviral nucleic acid is deficient for theexpression of the E1 A protein. The preferably cellular replicationsystem comprises a nucleic acid of a helper virus, whereby the nucleicacid of the helper virus contains a nucleic acid sequence which codesfor YB-1. In this connection the adenoviral nucleic acid or the nucleicacid of the helper virus may be present individually or separately asreplicable vectors.

The nucleic acids according to the present invention can be present asvectors. They are preferably viral vectors. In the case of the nucleicacids according to the present invention comprising adenoviral nucleicacids, the virus particle is in this case the vector. However, it isalso within the scope of the present invention that the nucleic acidsaccording to the present invention are present in a plasmid vector. Insuch a case the vector has elements which are responsible for or controlthe multiplication of the inserted nucleic acid (replication) andoptionally the expression of the inserted nucleic acid. Suitable vectorsand especially also expression vectors and elements are known to personsskilled in the art and are described for example in Grunhaus, A.,Horwitz, M. S., 1994, Adenoviruses as cloning vectors. In Rice, C.,editor, Seminars in virology, London: Saunders Scientific Publications,1992; 237-252.

The embodiment described above in which the various elements of thenucleic acid according to the invention do not necessarily have to becontained in only one vector, takes into account the aspect of theinvention which comprises the group of vectors. Correspondingly a groupof vectors consists of at least two vectors. Otherwise the same appliesto the vectors as has generally been stated herein with regard tovectors.

The adenoviruses according to the present invention are characterized bythe various nucleic acids disclosed herein and otherwise contain allelements known to persons skilled in the field which is also the casefor adenoviruses of the wild-type (Shenk, T.: Adenoviridae: The virusand their replication. Fields Virology, 3^(rd) edition, editor Fields,B. N., Knipe, D. M., Howley, P. M. et al., Lippincott-Raven Publishers,Philadelphia, 1996, chapter 67).

The agents according to the present invention, i.e. the nucleic acids,and vectors and groups of vectors, cells and adenoviruses and adenoviralreplication systems comprising the nucleic acids can be used for themanufacture of a medicament. As a result of the specific activities ofthe agents according to the present invention the medicament ispreferably used as such for the treatment or prophylaxis of tumourdiseases. These agents are basically suitable for all tumour diseases ortumours, and in particular also for tumours which contain p53 and thosein which p53 is absent. The term tumour encompasses both malignant andbenign tumours.

The medicament can be present in various formulations, preferably in aliquid form. The medicament also contains auxiliary substances such asstabilizers, buffers, preservatives and such like which are known to aperson skilled in the field of galenics.

In particular the medicament can also contain other pharmaceuticallyactive compounds. The type and the amount of these otherpharmaceutically active compounds will depend on the type of indicationfor which the medicament is used. If the medicament is used for thetreatment and/or prophylaxis of tumour diseases, cytostatic agents aretypically used such as cis-platinum and Taxol, Daunoblastin, Adriamycinand/or Mitoxantron.

The use of attenuated adenoviruses such as Onyx-015 which is deficientin the E1B-55 kDa protein, has relatively little chance of success whenused for viral oncolysis. The present inventor has surprisingly foundthat these attenuated viruses and especially also Onyx-015 can be usedwith a particularly high rate of success for tumours in which YB-1occurs in the cell nucleus independent of the cell cycle. YB-1 isnormally present in the cytoplasm and especially also in the perinuclearplasma. In the S phase of the cell cycle, YB-1 is located in the cellnucleus of normal and tumour cells. However, this is not sufficient toachieve a viral oncolysis using attenuated adenoviruses. Thecomparatively low effectiveness of attenuated adenoviruses such asOnyx-015 described in the prior art is also due to its erroneousapplication. In other words such systems and especially also Onyx-015can be used very effectively when the molecular biological prerequisitesfor viral oncolysis are present. In the case of especially Onyx-015these prerequisites are present in tumour diseases in which the cellshave a nuclear localisation of YB-1 independent of the cell cycle. Thisform of nuclear localisation may be due to the type of the tumour or maybe caused by the agents according to the invention described herein.Hence the present invention defines a new group of tumours or tumourdiseases and thus also of patients which can be treated with a high rateof success with the agents according to the present invention andespecially also with the attenuated adenovirus that has already beendescribed in the prior art and preferably with E1B-deficient, morepreferably E1B-55 kDa-deficient adenovirus and more preferably withOnyx-015.

Another group of patients that can be treated by using the adenovirusesdescribed in the prior art and especially those that are deficient inE1B such as ONYX-015, are those in which it is ensured that YB-1migrates or is transported into the nucleus by setting up certainconditions. The use of such adenoviruses in this group of patients isthus based on the finding that the induction of viral replication isbased on the nuclear localisation of YB-1 with subsequent binding to theE2 late promoter. The findings disclosed herein show that the adenovirusONYX-015 which is E1B-deficient, is not able to mediate transport of thecellular YB-1 into the nucleus. This is the reason for the limited useand success of ONYX-015 for such tumours which already have YB-1 in thenucleus. However, this is only the case for a very small group ofpatients. The localization of YB-1 in the nucleus can be induced byexternal stress or locally applied stress. This induction can forexample be achieved by irradiation, in particular UV irradiation, andadministration of cytostatic agents which have among others also beendisclosed herein, and hyperthermia. In connection with hyperthermia, itshould be noted that this can now be achieved very specifically and thusthe transport of YB-1 into the nucleus is also specific and consequentlythe requirements for replication of the adenovirus and thus for cell andtumour lysis are fulfilled (Stein U, Jurchott K, Walther W., Bergmann S.Schlag P M, Royer H D. Hyperthermia-induced nuclear translocation oftranscription factor YB-1 leads to enhanced expression of multidrugresistance-related ABC transporters. J. Biol. Chem. 2001,276(30):28562-9; Hu Z, Jin S, Scotto K W. Transcriptional activation ofthe MDR1 gene by UV irradiation. Role of NF-Y and Spl. J. Biol. Chem.2000 Jan. 28; 275(4):2979-85; Ohga T, Uchiumi T, Makino Y, Koike K, WadaM, Kuwano M, Kohno K. Direct involvement of the Y box binding proteinYB-1 in genotoxic stress-induced activation of the human multidrugresistance 1 gene. J. Biol. Chem. 1998, 273(10:5997-6000).

Hence the medicament according to the present invention would beadministered to such patients and groups of patients and would also besuitable for those in which transport of YB-1, especially in thecorresponding tumour cells, would be induced by suitable pretreatments.

Thus another aspect of the invention is also related to a method for thescreening of patients that can be treated with an attenuated adenovirussuch as Onyx-015 or in general with E1B-deficient, preferably E1B-55kDa-deficient adenovirus characterized by the following steps:

-   -   examining a sample of the tumour tissue and    -   determining whether YB-1 is located in the nucleus independent        of the cell cycle.

In this connection the sample of the tumour tissue can be obtained bypuncture or by surgery. Microscopic techniques and/orimmunohistoanalysis typically using antibodies are frequently used todetermine whether YB-1 is located in the nucleus independent of the cellcycle. YB-1 is detected using an agent which is selected from the groupcomprising antibodies against YB-1. The test for the nuclearlocalization of YB-1 and in particular independently of the cell cycleis known to a person skilled in the art. For example the localization ofYB-1 can be easily detected when screening tissue sections stained forYB-1. In this case the frequency of the occurrence of YB-1 in thenucleus already indicates that it is a cell cycle-independentlocalization in the nucleus. Another method for the cellcycle-independent detection of YB-1 in the nucleus is to stain for YB-1and determine whether YB-1 is located in the nucleus and to determinethe cell stage of the cells. This can also be carried out using suitableantibodies (double immunohistoanalysis).

The invention is further elucidated in the following on the basis of thefigures and examples from which further features, embodiments,applications and advantages may be taken.

FIG. 1 shows the basic molecular-genetic organization of the adenovirus;

FIG. 2 shows an overview of various nucleic acid and adenovirusconstructs according to the present invention and

FIG. 3 shows the result of a Northern Blot analysis.

FIG. 2 shows different nucleic acids and nucleic acid constructsaccording to the present invention. Thus FIG. 2.1A shows a recombinantadenoviral vector according to the present invention which isE1A-deficient and E1B-deficient but expresses the protein YB-1.

FIG. 2.1B shows a recombinant adenoviral vector according to the presentinvention which also expresses YB-1 but in which the E1A region isdeleted. Since E1A is responsible for the expression of E1B, E1B is notactivated or only to a reduced extent although the vector comprises thegene.

FIG. 2.2 shows a recombinant adenoviral vector according to the presentinvention which also expresses YB-1. In this case the gene E1B andE1B-55 kDa, respectively, is controlled by an external E1A-independentpromoter. Such a promoter can be the CMV, RSV or YB-1 promoter.

FIG. 2.3 shows another recombinant adenoviral vector according to thepresent invention which has a YB-1-independent E2 late promoter. This isachieved by completely removing the E2 late promoter or by specificallychanging the gene sequence within the promoter to which YB-1 binds (theso-called Y box).

FIG. 2.4 shows another recombinant adenoviral vector according to thepresent invention in which the E2 late as well as the E2 early promoterare deleted and are thus no longer functionally active. In the vectorshown schematically the two promoters are replaced by a tumour-specificor tissue-specific promoter as disclosed herein.

EXAMPLE 1 Importance of YB-1 for Adenoviral Replication

In order to prove that YB-1 controls the expression of E2 via the E2late promoter the following experimental approach was pursued.

Tumour cells (HeLa cells) were either infected with wild-typeadenovirus, an E1-minus adenovirus or with an E1-minus adenovirus whichexpresses YB-1 (AdYB-1) (in this connection K represents the control;non infected). The entire RNA was isolated after 24 h. Subsequently aNorthern blot analysis was carried out. The isolated RNA was thenseparated according to size by gel electrophoresis in aformaldehyde-agarose gel and blotted on a nylon membrane and fixed underUV. A cDNA fragment of 250 bases which is complementary to a sequencewhich is located between the E2 early promoter and the E2 late promoterwas used as a radioactively labelled probe. The probe was labelled withthe aid of the random prime labeling system from Amersham. An analysisof the films showed that an E2-specific signal is only present in cellsinfected with the wild-type adenovirus.

EXAMPLE 2 Importance of YB-1 for Adenoviral Replication

In order to prove that YB-1 controls the expression of E2 via the E2late promoter the following experimental approach was pursued which isbased on the protocol described in example 1.

The radioactive probe of example 1 was removed by boiling for 2 minutesin water and again hybridized with another cDNA probe. This probe islocated upstream of the E2 late promoter.

The analysis gave the following result: The cells infected withwild-type adenovirus as well as the cells infected with AdYB-1 have aclear signal and a specific E2 band. Hence YB-1 controls and activatesthe E2 region via the E2 late promoter.

EXAMPLE 3 Proof of the Specific Binding of YB-1 to the E2 Promoter

The experiment is based on the consideration that YB-1 as atranscription factor should bind to the Y box (CAAT sequence) within theE2 late promoter. In order to detect such a specific binding of YB-1 tothis promoter, a so-called EMSA analysis (electrophoretic mobility shiftassay) is carried out. For this the nuclear protein is isolated 24 hafter infecting the cells with wild-type adenovirus. Subsequently 1 —10μg protein and a short DNA fragment with a length of 30 to 80 baseswhich comprises the E2 late promoter sequence are incubated together for30 minutes at 37° C. This DNA fragment (oligo) is previouslyradioactively labelled with ³²P at the 5′ end using a kinase. Afterwardsa native polyacrylamide gel is used for separation. If the protein YB-1binds to a sequence on the oligo, a so-called shift results since theshort DNA fragment, which is radioactively labelled at the 5′ end,migrates in the gel due to the binding of YB-1 to the short DNA fragmentmore slowly than an unbound oligo. This shift can be abolished again assoon as a 100-fold excess of non-labelled oligo is added to the reactionmixture.

As a result it has been determined that YB-1 binds specifically to theE2 late promoter.

Competition experiments were carried out as a control. An excess ofnon-labelled E2 late promoter fragment was added to the reactionmixture. Subsequently a shift is no longer observed in the abovereaction mixture.

If reference is made to the prior art in the above description, it orits disclosed contents are incorporated herein by way of reference.

The features of the invention disclosed in the previous description, theclaims and the figures can be important individually as well as in anydesired combination for the realization of the invention in its variousembodiments.

1-46. (canceled)
 47. A nucleic acid comprising an adenoviral nucleicacid, characterized in that the nucleic acid comprises a nucleic acidsequence coding for YB-1.
 48. The nucleic acid of claim 47,characterized in that the adenoviral nucleic acid comprises a nucleicacid coding for E1-B.
 49. The nucleic acid of claim 48, characterized inthat the adenoviral nucleic acid comprises a nucleic acid coding forE4orf6.
 50. The nucleic acid of claim 48, characterized in that apromoter is provided which controls the expression of E1-B.
 51. Thenucleic acid of claim 49, characterized in that a promoter is providedwhich controls the expression of E4orf6.
 52. The nucleic acid of claim48, characterized in that E1B is the E1-B 55 kDa protein.
 53. Thenucleic acid of the claim 48, characterized in that the nucleic acidcodes for functionally inactive gene products E1 B and/or E1 and/or E3and/or E4.
 54. A nucleic acid comprising a nucleic acid coding for YB-1and a nucleic acid sequence mediating a nuclear transport of YB-1. 55.The nucleic acid of claim 54, characterized in that the nucleic acidsequence mediating the nuclear transport of YB-1 is selected from thegroup comprising signal sequences and transport sequences.
 56. A nucleicacid comprising an adenoviral nucleic acid, characterized in that theadenoviral nucleic acid comprises a tumour-specific promoter instead ofa functional E2 late promoter.
 57. A nucleic acid comprising anadenoviral nucleic acid, characterized in that the adenoviral nucleicacid comprises a tissue-specific promoter instead of a functional E2late promoter.
 58. The nucleic acid of claim 57, characterized in thatthe adenoviral nucleic acid contains a non-functional E2 early promoter.59. An adenoviral replication system comprising an adenoviral nucleicacid, whereby the adenoviral nucleic acid is deficient for theexpression of the E1A protein, and comprising a nucleic acid of a helpervirus, whereby the nucleic acid of the helper virus comprises a nucleicacid sequence which codes for YB-1.
 60. The adenoviral replicationsystem of claim 59, characterized in that the adenoviral nucleic acidand/or the nucleic acid of the helper virus is present as a replicablevector.
 61. A vector, preferably an expression vector, comprising anucleic acid of claim 47, 54, 56 or
 57. 62. A group of vectorscomprising of at least two vectors, whereby the overall group of vectorscontains an adenoviral replication system of claim
 60. 63. The group ofvectors of claim 62, characterized in that each component of theadenoviral replication system is located on a suitable vector,preferably an expression vector.
 64. The group of vectors of claim 62,characterized in that at least two components of the adenoviralreplication system are located on a vector of the group of vectors. 65.An adenovirus comprising a nucleic acid of claim 47, 54, 56 or
 57. 66.The adenovirus of claim 65 comprising a capsid.
 67. A cell comprising anucleic acid of claim 47, 54, 56 or 57 and/or an adenoviral replicationsystem of claim 60 and/or a vector of claim 61 and/or a group of vectorsof claim 62 and/or an adenovirus of claim
 65. 68. Use of a nucleic acidof claim 47, 54, 56 or 57 and/or an adenoviral replication system ofclaim 60 and/or a vector of claim 61 and/or a group of vectors of claim62 and/or an adenovirus of claim 65 and/or a cell claim 67 for themanufacture of a medicament.
 69. The use of claim 68, characterized inthat the medicament is used for the treatment and/or prophylaxis oftumour diseases.
 70. The use of claim 68, characterized in that themedicament additionally contains a pharmaceutically effective compound.71. The use of claim 70, characterized in that the pharmaceuticallyeffective compound is selected from the group comprising cytostaticagents, whereby the cytostatic agents are preferably selected from thegroup comprising cis-platinum, Taxol, Daunoblastin, Adriamycin andMitoxantron.
 72. Use of a nucleic acid of claim 47, 54, 56 or 57 and/oran adenoviral replication system of claim 60 and/or a vector of claim 58and/or a group of vectors of claim 62 and/or an adenovirus of claim 65and/or a cell of claim 67 for the manufacture of a medicament for thetreatment and/or prophylaxis of tumour diseases, whereby YB-1 is locatedin the nucleus of the tumour cells independent of the cell cycle. 73.The use of claim 72, characterized in that the nucleic acid codes for anadenoviral nucleic acid and that the adenoviral nucleic acid isE1B-deficient, and in particular E1B 55 kDa-deficient.
 74. The use ofclaim 72, characterized in that the adenovirus is E1B-deficient, and inparticular E1B 55 kDa-deficient.
 75. The use of claim 72, characterizedin that the nucleic acid codes for an adenoviral nucleic acid and thatthe adenoviral nucleic acid codes for E1B, and codes in particular forE1B 55 kDa.
 76. The use of claim 72, characterized in that theadenovirus expresses E1B, and expresses in particular E1B 55 kDa. 77.The use of claims 72, characterized in that the adenoviral nucleic acidcodes for E1 A.
 78. The use of claims 72, characterized in that theadenovirus expresses E1A.
 79. The use of claim 76, characterized in thatthe tumour and/or the tumour disease is selected from the groupcomprising p53-positive tumours, p53-negative tumours, malignanttumours, benign tumours and combinations thereof.
 80. A method for thescreening of patients who can be treated with an E1B-deficient,preferably E1B 55 kDa-deficient adenovirus characterized by thefollowing steps: examining a sample of the tumour tissue and determiningwhether YB-1 is located in the nucleus independently of the cell cycle.81. The method of claim 80, characterized in that the tumour tissue isexamined using antibodies against YB-1.